Zhou Feng-Quan
Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine.
J Vis Exp. 2012 Aug 17(66):4141. doi: 10.3791/4141.
It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS(1,2). In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries(3). Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved(4). Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth(5-7). Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection(6,8). By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons(9).
众所周知，由于支持轴突生长的内在能力减弱以及成熟中枢神经系统（CNS）中的不利环境，中枢神经系统（CNS）中的成熟神经元在受伤后无法再生其轴突(1,2)。相比之下，外周神经系统（PNS）中的成熟神经元在受伤后很容易再生(3)。众所周知，成年背根神经节（DRG）神经元在周围神经损伤后能强劲再生。每个DRG神经元从细胞体长出一根轴突，该轴突分支为两个轴突分支：一个支配外周靶标的外周分支和一个延伸到脊髓的中枢分支。DRG外周轴突损伤会导致大量轴突再生，而脊髓中的中枢轴突在损伤后再生较差。然而，如果外周轴突损伤发生在脊髓损伤之前（这一过程称为预处理损伤），中枢轴突的再生会得到极大改善(4)。此外，DRG神经元的中枢轴突与脊髓中下行皮质脊髓轴突共享相同的不利环境。综合来看，人们推测可以利用控制成年DRG神经元轴突再生的分子机制来增强中枢神经系统轴突再生。因此，成年DRG神经元现在被广泛用作研究再生轴突生长的模型系统(5-7)。在这里，我们描述了一种成年DRG神经元培养方法，可用于体外轴突再生的遗传学研究。在这个模型中，成年DRG神经元通过电穿孔介导的基因转染进行基因操作(6,8)。通过用DNA质粒或si/shRNA转染神经元，这种方法能够进行功能获得和功能丧失实验，以研究任何感兴趣的基因在成年DRG神经元轴突生长中的作用。当用si/shRNA转染神经元时，靶向的内源性蛋白通常在培养3-4天后耗尽，而在此期间已经发生了强劲的轴突生长，这使得功能丧失研究效果较差。为了解决这个问题，这里描述的方法在转染后包括一个重悬和重新铺板步骤，这使得轴突能够在没有靶向蛋白的情况下从神经元重新生长。最后，我们提供了一个使用这个体外模型研究轴突再生相关基因c-Jun在介导成年DRG神经元轴突生长中的作用的例子(9)。
